This invention relates to a method of measuring antithrombin activity.
Blood coagulation or clotting results when a series of inactive enzymes in blood are activated to generate, at the end of the cascade, a clot at the site of the wound. The intrinsic blood coagulation pathway is activated upon contact with the surface of a foreign matter which initiates the sequential activation of factors XII to XIIa, prekallikrein to kallikrein, kininogen to kinin, XI to XIa, IX to IXa, X to Xa, and II (prothrombin) to IIa (thrombin). Tissue thromboplastin initiates the extrinsic blood coagulation pathway by activation of factor X, which in turn results in the activation of prothrombin to form thrombin. In both pathways, the final enzyme in the cascade is thrombin, a serine protease which cleaves the soluble protein fibrinogen to form fibrin. Fibrin molecules crosslink to form a clot which reduces the flow of blood from the wound.
Antithrombin or antithrombin III (AT) is an important regulator of blood coagulation. AT, which is produced in the liver, is a serine protease inhibitor with a molecular weight of approximately 60,000 Daltons and circulates in the blood at a concentration of 150 to 200 micrograms per milliliter, or 2.5 to 3.4 micromoles per liter. AT has a broad specificity, and inhibits most of the coagulation factors involved in the intrinsic and the extrinsic pathways and is the principle regulator of thrombin. The inhibition of most coagulation enzymes by AT is significantly augmented in the presence of heparin.
AT deficiency is associated with an increased risk of thrombosis. The condition may be congenital, or acquired as a result of underlying conditions such as liver disease, kidney disease or disseminated intravascular coagulation. AT deficiency may be related to reduced levels of AT or reduced AT activity. For example, in congenital AT deficiency type II, the AT concentration is normal but the activity is reduced due to the presence of a dysfunctional AT. Successful clinical diagnosis and management of patients with AT deficiency therefore demands a specific, sensitive and a simple laboratory assay of the AT activity.
Current methods to determine AT deficiency can be divided into three classes: immunoassays, amidolytic-based activity assays and clot-based activity assays.
Immunoassay techniques measure the concentration of AT in a sample through methods such as radial immunodiffusion, nephelometry and enzyme-linked immunosorbant assays (ELISAs). These assays are very specific and quite sensitive, but can be time-consuming to perform. As well, concentration measurements of AT do not always correlate with AT activity levels since inactive forms of AT or AT-enzyme complexes may still exhibit immunoreactivity in these assays. This may lead to inappropriately high test results for some patients with reduced AT activity as in the case of type II deficiency.
Amidolytic-based activity assays work on the principle of incubating a fixed quantity of a single purified enzyme, usually thrombin or factor Xa, with a diluted test sample and heparin. The residual enzyme activity is measured by determining the endpoint or kinetic rate of cleavage of synthetic chromogenic or fluorogenic substrates. These types of assays are currently the most widely used methods to determine AT activity levels. However, these assays tend to be susceptible to interference from other coagulation inhibitors such as xcex12-macroglobulin, heparin cofactor II and xcex11-antitrypsin. As well, measurements of AT activity vary depending on which purified enzyme is used for the assay. Costs for these assays can be fairly high due to the use of purified enzymes and synthetic substrates, and the requirement for a spectrophotometer or high-end coagulation analyser to detect the reaction endpoint.
Clot-based activity assays may be performed as either two-stage or one-stage assays. The two-stage assays involve incubating a fixed quantity of purified enzyme, such as thrombin, with defibrinated test serum or plasma. Residual enzyme activity is measured by determining clotting activity upon the addition of plasma or purified fibrinogen instead of determining the amidolytic activity as described above. Drawbacks of these assays include artifactual reduction of AT levels if heat denaturation is used to defibrinate the plasma. Also, these methods tend to be cumbersome and labour intensive, time-consuming, and susceptible to interference by other progressive coagulation inhibitors.
A one-stage clot-based assay is described in U.S. Pat. No. 5,093,237 to Enomoto. In this assay, the test specimen is mixed with AT-free plasma containing the extrinsic coagulation factors, heparin and a prothrombin time measuring reagent and the coagulation time resulting from the activation of the extrinsic coagulation pathway is measured. The prothrombin time test is known to be extremely insensitive to heparin-enhanced inhibition by AT and the Enomoto assay discloses the use of a high concentration of heparin (12 U/ml). This is far above the optimal concentration of heparin for AT and at such high concentrations, it is known that the efficiency of inhibition by AT is reduced. Moreover, while Enomoto discloses that the extrinsic coagulation reaction is utilized to avoid the many potential errors in the intrinsic reaction pathway, the assay generates only two enzymes, activated Factor X and thrombin, upon which AT can exert its inhibitory effect and does not best reflect the full spectrum of in vivo physiological AT activity.
It is apparent therefore, that there remains to be developed a sensitive, specific yet simple clot-based laboratory assay for AT activity.
The present invention provides a method for measuring AT activity in samples containing AT, such as in a patient plasma sample. The method of the invention includes the step of mixing a test sample with an AT-deficient substrate plasma, an activator of the contact phase of the intrinsic coagulation pathway and a phospholipid. The AT-deficient plasma contains the enzymes of the intrinsic coagulation pathway and may also contain an AT augmenting compound, such as heparin. The AT augmenting compound if not present in the substrate plasma is added separately to the test sample. Following addition of calcium ions to the mixture, the coagulation time is measured. By comparing the coagulation time to a reference standard, the AT activity level of the test sample can be determined.
In another aspect, the invention provides a kit for measuring AT activity which kit includes an AT-deficient substrate plasma, an AT augmenting compound, an APTT reagent and a calcium salt solution.